Serotonin signals through postsynaptic Gαq, Trio RhoGEF, and diacylglycerol to promote C. elegans egg-laying circuit activity and behavior

Activated Gαq signals through Phospholipase-Cβ (PLCβ) and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to neurotransmitters like serotonin remains poorly understood. We used the egg-laying behavior circuit of C. elegans to determine whether PLCβ and Trio mediate serotonin and Gαq signaling through independent or related biochemical pathways. Our genetic rescue experiments suggest that PLCβ functions in neurons while Trio RhoGEF functions in both neurons and the postsynaptic vulval muscles. While Gαq, PLCβ, and Trio RhoGEF mutants all fail to lay eggs in response to serotonin, optogenetic stimulation of the serotonin-releasing HSN neurons restores egg laying only in PLCβ mutants. PLCβ mutants showed vulval muscle Ca2+ transients while strong Gαq and Trio RhoGEF mutants had little or no vulval muscle Ca2+ activity. Treatment with phorbol 12-myristate 13-acetate (PMA) that mimics 1,2-diacylglycerol (DAG), a product of PIP2 hydrolysis, rescued egg-laying circuit activity and behavior defects of Gαq signaling mutants, suggesting both Phospholipase-C and Rho signaling promote synaptic transmission and egg laying via modulation of DAG levels. DAG activates effectors including UNC-13, however we find that phorbol esters, but not serotonin, stimulate egg laying in unc-13 and PLCβ mutants. These results support a model where serotonin signaling through Gαq, PLCβ, and UNC-13 promote neurotransmitter release, and that serotonin also signals through Gαq, Trio RhoGEF, and an unidentified, PMA-responsive effector to promote postsynaptic muscle excitability. Thus, the same neuromodulator serotonin can signal in distinct cells and effector pathways to coordinate activation of a motor behavior circuit.


Introduction
Plasmids KG#68 (15 ng/µl; pan-neuronal GFP) or pKMC166 (15 ng/µl; pan-muscle mCherry) 141 alone or with KG#281 (50ng/µl; pan-neuronal unc-73e) and pPD3 (50ng/µL; pan-muscle unc-142 73e) were injected into KG1278 unc-73(ce362) I, generating five independent mCherry(+), 143 GFP(+) transgenic lines for behavior experiments from which a single transgenic line from each 144 was kept: MIA372 unc-73(ce362); keyEx64 expressing mCherry (muscles) and GFP (neurons) 145 only and MIA373 unc-73(ce362); keyEx65 expressing GFP (neurons), mCherry (muscles) and 146 TrioRhoGEF-E (both neurons and muscles). in each well were counted. Since egg-laying defective animals sometimes release one or two 235 eggs/L1 larvae when they are first picked into the well in response to mechanical stimulation, 236 animals were only recorded as responding if they laid 3 or more progeny. For Ca 2+ imaging, 237 NGM plates containing either PMA or ethanol solvent were prepared as described (Reynolds et 238 al., 2005). Age-matched adult worms from each genotype were placed on separate PMA or control NGM plates at room temperature for 1.5 hours. An agar chunk was then placed between 240 two glass coverslips for Ca 2+ activity recording as described (Ravi et al., 2018b All the data, reagents, and strains used in this study are available from the corresponding author 291 upon request.

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Trio RhoGEF acts in both neurons and muscles to drive egg-laying behavior 294 Prior studies have shown that Gaq signaling (Fig. 1A)    RhoGEF-E and measured egg accumulation in these animals. We observed a modest, but  357 whose release of ACh is regulated by serotonin and G protein signaling .

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To test how serotonin promotes egg laying via Gaq, we measured the egg-laying response to    (Fig. 5A). We measured the egg-laying responses of wild-type animals and Gaq signaling 485 mutants to Phorbol 12-myristate 13-acetate (PMA). As shown in Figure 5B, 10 µM PMA treatment strongly stimulated egg laying in wild-type animals and all mutants with reduced Gaq 487 signaling (≥80% animals laying eggs). Like serotonin (Fig. 2B), PMA also rescued egg laying in 488 egl-1(n986dm) mutant animals lacking the HSNs (Fig. 5B)  We next imaged how PMA affected vulval muscle Ca 2+ activity. We performed 10-minute 512 GCaMP5 Ca 2+ recordings of wild-type or Gaq signaling mutants after two hours of exposure to 513 10 µM PMA (Movies 1-3). Quantitation of Ca 2+ transients showed that PMA significantly 514 increased vulval muscle Ca 2+ activity in wild-type animals to 10±1.6 transients per minute from 515 an average 2±0.9 transients per min in vehicle-treated, wild-type animals (Fig. 5C-D  behavior through activation of UNC-13 or PKC, we tested whether mutants lacking these 532 proteins still have a robust serotonin and/or PMA egg-laying response (Fig. 6). Mutants that 533 eliminate axonal and synaptic UNC-13 show reduced egg laying, accumulating an average of 534 22 eggs compared to 15 seen in wild-type animals (Fig. 6A), like PLCβ mutants but significantly fewer than the >30 eggs that accumulate in Gaq and Trio RhoGEF mutants (Fig. 1C-I). unc-13 536 mutants also resemble PLCβ mutants in their egg-laying response to serotonin and PMA. Egg 537 laying in unc-13 mutants was stimulated by PMA but was resistant to serotonin (Fig. 6B). While 538 the serotonin resistance we observed for unc-13 mutants after one hour differs from that seen 539 by Bastiani et al., 2003 at 90 minutes, we saw similar differences for the PLCβ mutants (Table   540 3). These results show that serotonin promotes egg laying through a PLCβ and UNC-13-541 dependent pathway that may be distinct from the PMA-stimulated pathway.

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To determine whether Gaq and Trio RhoGEF signaling through the PMA-responsive  (Fig. 6C). Animals bearing predicted null mutants of novel and conventional PKCs such as 551 nPKCδ/θ (TPA-1) and cPKCα/β (PKC-2) orthologs also show no significant differences in egg 552 accumulation (Fig. 6C), suggesting that, unlike loss of Gaq or Trio RhoGEF signaling, disruption 553 of individual PKC signaling pathways do not strongly affect egg-laying behavior. We next tested 554 whether PKC mediates the egg-laying response to PMA. All the PKC single mutant animals laid 555 eggs in response to PMA except for pkc-1(nj1) (Fig. 6C). The nj1 allele is predicted to be a

566
In this study we explored the cellular and molecular specificity of Gaq effector signaling as it 567 regulates egg-laying circuit activity and behavior using molecular genetics, optogenetics, 568 pharmacology, and Ca 2+ imaging techniques. We found that Gaq effectors PLCβ and Trio 569 RhoGEF differentially act in neurons and muscles to promote synaptic transmission and egg-570 laying behavior, supporting a working model where Gaq signals through Trio RhoGEF in both 571 neurons and muscles while PLCβ functions outside of HSN to promote egg laying (Fig. 7).